Sample to Sequence

ABSTRACT

Method and sample vessels are provided for amplification and sequencing of nucleic acids in a sample.

BACKGROUND

There are a variety of different applications in which asample-to-sequence result in a short period of time would have greatutility. These applications may include rapid, near point-of-care orlaboratory, identification of mutations that confer antibioticresistance, sequencing of viral genes, identification of tissue forforensic purposes, tissue typing for organ transplantation, andidentification of alleles related to rates of drug metabolism, forexample.

For example, for bacterial pathogens implicated in sepsis, it is usefulto know whether the bacteria carry extended spectrum beta-lactamases(ESBLs) that make them resistant to extended spectrum cephalosporins.These ESBLs result from mutations in the TEM-1, TEM-2, SHV, and otherbeta lactamase genes. There are numerous other genes that can confer anantibiotic resistance phenotype, whose expression or activity ismodulated by point mutations in the coding or regulatory regions ofthese genes. While single known point mutations are easy to assay usingstandard PCR techniques, mutations in genes that have multiple allelesmay be more difficult to identify.

Likewise there are numerous instances in which the sequence of a viralgene can indicate both the presence of that virus in a human sample andwhether that virus is resistant to antiviral compounds, and thusindicating what treatment should be started, continued, or stopped. Thisis true for Hepatitis C, Hepatitis B, and HIV. While time-to-result isnot quite as pressing for these chronic viral infections, time is of theessence for other viral infections, such as influenza. With influenzaand other acute viral infections, there are an increasing number ofwell-characterized point mutations that confer resistance to the FDAapproved neuraminidase inhibitors. Ideally, treatment with aneuraminidase inhibitor should start as soon as possible after the virusis detected, but it is important that the correct inhibitor be used.

In addition to pathogen detection and identification, there are severalinstances in which determining the sequence of a human gene or genesquickly is important. These include identification of human tissue forforensic purposes, illustratively by mapping the length of Short TandemRepeats (STRs). This identification is currently performed by sizing PCRamplicons, but it can also be done by sequencing. A quicksample-to-sequence method would be helpful in many such cases.

Another example is HLA typing an organ for transplantation. Organs to bedonated may come from a recently deceased individual. It is important toget the organ delivered quickly to the transplant recipient that has thebest MHC match to the donor. The most accurate way to do this is tosequence the MHC genes that govern transplant rejection and then matchthese sequences to a national registry of recipient MHC types. A quicksample-to-sequence method would be helpful in many organ donation cases.

Yet another example is identification of cytochrome P450 alleles thatdetermine rates of drug metabolism. It is known that cytochrome P450enzymes in the liver metabolize foreign compounds for excretion.Multiple proteins in this family are known, and different alleles ofeach are present in different populations and individuals. For example,some people carry alleles for enzymes that metabolize warfarin very fastand some metabolize warfarin much more slowly. Knowing a patient'sgenotype permits a doctor to decide how much warfarin or other suchdrugs to administer to achieve a certain level of drug in the bloodstream.

Other non-limiting examples where sequencing could be useful includescancer genes, as well as other infectious agents such as tuberculosis.Many other examples are possible, as are uses both in clinicaldiagnostics and in other fields.

Polymerase chain reaction (PCR) is a technique widely used in molecularbiology. It derives its name from one of its key components, a DNApolymerase used to amplify a piece of DNA by in vitro enzymaticreplication. As PCR progresses, the DNA generated (the amplicon) isitself used as a template for replication. This sets in motion a chainreaction in which the DNA template is exponentially amplified. With PCR,it is possible to amplify a single or few copies of a piece of DNAacross several orders of magnitude, generating millions or more copiesof the DNA piece. PCR employs a thermostable polymerase, dNTPs(deoxynucleotide triphosphates), and a pair of primers(oligonucleotides). Existing PCR techniques and other amplificationmethods may be combined with next generation sequencing (NGS) to providequick sample-to-sequence results.

Traditional nucleic acid sequencing techniques employ either chemicalcleavage at a specific base (Maxim-Gilbert method) or chain terminationusing dideoxynucleotides (Sanger sequencing). Next-generation sequencinginvolves high-throughput sequencing technologies some of whichparallelize the sequencing process, producing thousands to millions ofsequences concurrently, often detecting as each nucleotide is added toeach individual strand. Various next-generation systems are currentlyavailable.

BRIEF SUMMARY

In one aspect of the present disclosure, methods for amplification andsequencing of nucleic acids in a sample. Illustrative methods maycomprise the steps of placing the sample in an amplification chamber,wherein the amplification chamber is configured for amplifying aplurality of individual nucleic acids that may be present in the sample,subjecting the amplification chamber to amplification conditions, movingthe sample to an array of second-stage amplification wells, eachsecond-stage amplification well configured for further amplifying oneindividual nucleic acid that may be present in the sample, such that aportion of the nucleic acids are moved to each of the additionalsecond-stage amplification wells, performing second-stage amplificationin the additional second-stage amplification wells to generate anamplicon in each second-stage amplification well if the individualnucleic acid is present in the sample, and subjecting at least aplurality of the wells to sequencing conditions.

In some embodiments, the systems and methods described herein includemethods for amplification and sequencing of nucleic acids in a samplewith the methods comprising the steps of placing the sample in afirst-stage amplification chamber, where the first-stage amplificationchamber is configured for amplifying a plurality of individual nucleicacids that may be present in the sample, subjecting the first-stageamplification chamber to amplification conditions, moving the sample toan array of second-stage amplification wells with each second-stageamplification well configured for further amplifying one individualnucleic acid that may be present in the sample, such that a portion ofthe sample is moved to each of the second-stage amplification wells,performing second-stage amplification in the second-stage amplificationwells to generate an amplicon in each second-stage amplification well ifthe individual nucleic acid is present in the sample, and subjectingcontents of at least one second-stage amplification well to sequencingconditions. In some cases, each of the additional second-stageamplification wells has a primer tethered thereto, and sequencing canoccur in the second-stage amplification wells. In other cases, all stepsare performed in a single container and the last two steps performed inthe wells. In yet other cases, the container is provided with one ormore sealable ports, with the sealable ports providing the only accessfrom an exterior of the container to the first-stage amplificationchamber and the array of second-stage amplification wells, such thatwhen the one or more sealable ports are sealed, the container is fullyclosed. In some cases, sequencing can be performed in a separate samplevessel. In other cases, all the steps are performed in about 5 hours orless. In yet other cases, all second-stage amplification wells aresubjected to sequencing conditions. In yet other cases, the methods canfurther comprise the step of detecting whether the amplicon has beengenerated in each second-stage amplification well to generate a positivecall for each second-stage amplification well where the amplicon hasbeen generated. In some instances, sequencing is performed only on thosesecond-stage amplification wells where there is the positive call. Inother instances, the first-stage amplification chamber comprises aplurality of first-stage pairs of primers, each of the first-stage pairsof primers configured for amplifying one of the plurality of individualnucleic acids that may be present in the sample, and wherein each of thesecond-stage amplification wells comprises a pair of second-stageprimers configured for amplifying one of the plurality of individualnucleic acids that may be present in the sample. In yet other instances,at least one of each of the pair of second-stage primers is nestedwithin its corresponding first-stage primer.

In some embodiments, the systems and methods described herein includemethods for amplification and sequencing of nucleic acids in a samplethat comprise the steps of placing the sample in an amplificationchamber, where the amplification chamber is configured for amplifying aplurality of individual nucleic acids that may be present in the sample,subjecting the amplification chamber to amplification conditions, movingthe sample to an array of spots, each spot comprising a set ofsecond-stage amplification primers tethered thereto, each set ofsecond-stage amplification primers configured for further amplificationof one of the individual nucleic acids, performing second-stageamplification on the array to generate an amplicon at each of the spotsif that individual nucleic acid is present in the sample, and subjectingat least one of the spots to sequencing conditions. In some instances,all steps are performed in a single container. In other instances, thecontainer is provided with one or more sealable ports, the sealableports providing the only access from an exterior of the container to theamplification chamber and the array of second-stage amplification wells,such that when the one or more sealable ports are sealed, the containeris fully closed. In yet other instances, the array has an inlet channeland an outlet channel and opening and closing the inlet channel andoutlet channel controls flow of fluid across the array. In some cases,closure of the outlet channel while fluid enters the inlet channelresults in a bubble of fluid over the array, and agitation of the bubblehomogenizes the fluid. In other cases, the methods further comprise thestep of detecting whether the amplicon has been generated at each spotto generate a positive call for each well where the amplicon has beengenerated. In yet other cases, the number of spots totals no more than512 spots. In some instances, the number of spots totals no more than256 spots. In other instances, the set comprises both of the pair ofsecond-stage primers. In yet other instances, second-stage amplificationis performed in the presence of a polymerase having 3′ to 5′ exonucleaseactivity. In some cases, the set comprises one of the pair ofsecond-stage primers. In other cases second-stage amplification isperformed in the presence of a solution comprising a second of each ofthe pairs of second-stage primers. In yet other cases, the sample istreated with a polymerase having 3′ to 5′ exonuclease activitysubsequent to subjecting the amplification chamber to amplification. Insome instances, all of the second of the pairs of second-stage primersare provided with a 5′ sequence corresponding to a universal primer. Inother instances, the solution further comprises the universal primer. Inyet other instances, all of the second of the pairs of second-stageprimers are provided at a concentration no more than about 1/10th theconcentration of the universal primer. In some cases, the sample is notdiluted between the steps of subjecting the amplification chamber toamplification conditions and performing the second stage amplification.In other cases, at least one of the pair of second-stage primers has anadditional sequence corresponding to a universal primer, and sequencingis performed by synthesis using the universal primer.

In some embodiments, the systems and methods described herein includemethods for amplification and sequencing of nucleic acids that may bepresent in a sample, comprising the steps of amplifying in the presenceof a plurality of outer primer pairs, each outer primer pair configuredfor amplifying one of the nucleic acids and in the presence of at leastone inner primer for each of the nucleic acids, to generate an ampliconfor each of the nucleic acids present in the sample, and subjecting theamplicons to sequencing conditions. In other embodiments, the pluralityof outer primer pairs is located in one reaction chamber and the innerprimers are located in a separate reaction chamber. In yet otherembodiments, the plurality of outer primer pairs and the inner primersare located in a single reaction chamber.

In some embodiments, the systems and methods described herein includemethods for amplifying nucleic acids in a sample comprising the steps ofplacing the sample in a first-stage amplification chamber, where theamplification chamber comprises a first pair of primers configured foramplifying a nucleic acid that may be present in the sample, subjectingthe amplification chamber to amplification conditions to generate anamplicon, moving a first portion of the sample to a first second-stageamplification zone comprising a first plurality of sets of second-stageamplification primers, each set of second-stage amplification primersconfigured to amplify different non-overlapping regions of the amplicon,moving a second portion of the sample to a second second-stageamplification zone comprising a second plurality of sets of second-stageamplification primers, each set of second-stage amplification primersconfigured to amplify different non-overlapping regions of the amplicon,and performing second-stage amplification on the first second-stageamplification zone to generate first second-stage amplicons and on thesecond second-stage amplification zone to generate second second-stageamplicons, where at least some of the first second-stage ampliconsoverlap at least some of the second second-stage amplicons. In otherembodiments, the methods further comprise subjecting at least one of theamplicons to sequencing conditions.

In some embodiments, the systems and methods described herein includemethods for amplification and sequencing of nucleic acids in a samplecomprising the steps of placing the sample in an amplification chamber,wherein the amplification chamber comprises a first forward primer, asecond forward primer nested within the first forward primer, and areverse primer, wherein the second forward primer is tethered to astructure and subjecting the amplification chamber to amplificationconditions. In other embodiments, the methods further comprisesubjecting the amplification chamber to sequencing conditions.

In some embodiments, the systems and methods described herein includecontainers for amplification and sequencing of a plurality of nucleicacids comprising a first-stage amplification chamber, the first-stageamplification chamber comprising a plurality of first-stage pairs ofprimers, each first-stage pair of primers configured for amplificationof one of the plurality of nucleic acids to generate first-stageamplicons and a second-stage amplification chamber, the second-stageamplification chamber comprising a plurality of second-stage primerpairs, each second-stage pair of primers configured for amplification ofat least a portion of a sequence of one of the first stage-amplicons togenerate second-stage amplicons, where the second-stage amplificationchamber is further configured for sequencing the second-stage amplicons.In some cases, at least one member of each of the second-stage primerpairs is nested within a corresponding first-stage primer. In othercases, at least one member of each of the second-stage primer pairs istethered to a support. In yet other cases, the at least one member ofeach of the second-stage primer pairs is tethered to the support by the5′ end. In some instances, the second-stage amplicons comprise singlemolecular species. In other instances, the containers further compriseone or more sealable ports, the one or more sealable ports providing theonly access from an exterior of the container to the first amplificationchamber and the second-stage amplification chamber, such that when theone or more sealable ports are sealed, the container is fully closed. Inyet other instances, the second-stage amplification chamber comprises aninlet channel and an outlet channel and opening and closing the inletchannel and the outlet channel controls flow of fluid across thesecond-stage amplification chamber. In some cases, the containersfurther comprise a lysis chamber configured to receive a sample and toprepare a lysed sample. In other cases, the containers further comprisea nucleic acid extraction chamber configured to receive the lysed sampleand to extract nucleic acids from the lysed sample. In some instances,the second-stage amplification chamber comprises an array of wells, eachwell configured to carry out a second-stage amplification reaction. Inother instances, the second-stage amplification chamber comprises anarray of spots, each spot comprising second-stage primer pairs, and eachspot configured to carry out a second-stage amplification reaction. Inyet other instances, at least one of the second-stage primers istethered to the spot. In some cases, the number of spots totals no morethan 512 spots. In other cases, the number of spots totals no more than256 spots.

In some embodiments, the systems and methods described herein includemethods for two-step amplification and sequencing of a plurality ofnucleic acids that may be in a sample comprising amplifying the nucleicacids in a first mixture comprising a plurality of first-stage pairs ofprimers, each first-stage pair of primers configured for amplificationof one of the plurality of nucleic acids to generate a first-stageamplicon for each of the nucleic acids that are present in the sample,amplifying the first-stage amplicons using a plurality of second-stagepairs of primers, each second-stage pair of primers configured foramplification of one of the first-stage amplicons, wherein at least oneof each pair of second-stage primers is nested within its correspondingfirst-stage pair of primers, to generate a single molecular species foreach of the nucleic acids that are present in the sample, and sequencingthe single molecular species that are generated. In other embodiments,the steps of amplifying and sequencing are performed in a singlereaction chamber. In yet other embodiments, at least one of each pair ofsecond-stage primers is tethered to a solid support. In someembodiments, the steps of amplifying and sequencing are performed indifferent reaction vessels. In other embodiments, no filter is usedbetween the steps of amplifying and sequencing to identify the correctamplicon.

Additional features and advantages of the embodiments of the inventionwill be set forth in the description which follows or may be learned bythe practice of such embodiments. The features and advantages of suchembodiments may be realized and obtained by means of the instruments andcombinations particularly pointed out in the appended claims. These andother features will become more fully apparent from the followingdescription and appended claims, or may be learned by the practice ofsuch embodiments as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the invention can be obtained, a moreparticular description of the invention briefly described above will berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 shows an illustrative pouch for use in one embodiment in thisdisclosure.

FIGS. 2A-B illustrate steps used for amplification and sequencing in asingle sample well.

FIG. 3 is a partially exploded view of an illustrative second-stage ofthe pouch of FIG. 1, configured for sequencing subsequent toamplification.

FIG. 4 is similar to FIG. 1, but showing a different embodiment for thesecond-stage array.

FIGS. 5A-5C are similar to FIGS. 2A-2B, except showing bridgeamplification.

FIGS. 6A-6E are similar to FIGS. 5A-5C, except showing a differentembodiment for use with the devices described herein.

FIGS. 7A-C show an embodiment for sequencing multiple amplificationproducts generated from a single first-stage amplicon. FIG. 7A showsoverlapping second-stage amplicons, while FIGS. 7A-7B shows one way ofdividing second-stage amplification into second-stage amplificationreactions.

FIGS. 8A-8C are similar to FIGS. 5A-5C, except showing an alternativeprimer embodiment.

DETAILED DESCRIPTION

Before describing example implementations in detail, it is to beunderstood that this disclosure is not limited to parameters of theparticularly exemplified systems, methods, apparatus, products,processes, compositions, and/or kits, which may, of course, vary. It isalso to be understood that the terminology used herein is only for thepurpose of describing particular implementations of the presentdisclosure, and is not necessarily intended to limit the scope of thedisclosure and/or invention in any manner Thus, while the presentdisclosure will be described in detail with reference to specificconfigurations, the descriptions are illustrative only and are not to beconstrued as limiting the scope of the claimed invention. For instance,certain implementations may include fewer or additional components thanthose illustrated in the accompanying drawings and/or described in thewritten description. Furthermore, various modifications can be made tothe illustrated configurations without departing from the spirit andscope of the invention as defined by the claims. Thus, while variousaspects, embodiments, and/or implementations of the invention aredescribed and/or disclosed herein, other aspects, implementations, andembodiments are also contemplated.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure pertains. While a number ofmethods and materials similar or equivalent to those described hereincan be used in the practice of the present disclosure, only certainexemplary materials and methods are described herein.

Various aspects of the present disclosure, including devices, systems,methods, etc., may be illustrated with reference to one or moreexemplary implementations. As used herein, the terms “exemplary” and“illustrative” mean “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other implementations disclosed herein. In addition, reference toan “implementation” or “embodiment” of the present disclosure orinvention includes a specific reference to one or more embodimentsthereof, and vice versa, and is intended to provide illustrativeexamples without limiting the scope of the invention, which is indicatedby the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a tile” includes one, two, or more tiles. Similarly,reference to a plurality of referents should be interpreted ascomprising a single referent and/or a plurality of referents unless thecontent and/or context clearly dictate otherwise. Thus, reference to“tiles” does not necessarily require a plurality of such tiles. Instead,it will be appreciated that independent of conjugation; one or moretiles are contemplated herein.

As used throughout this application the words “can” and “may” are usedin a permissive sense (i.e., meaning having the potential to), ratherthan the mandatory sense (i.e., meaning must). Additionally, the terms“including,” “having,” “involving,” “containing,” “characterized by,”variants thereof (e.g., “includes,” “has,” and “involves,” “contains,”etc.), and similar terms as used herein, including the claims, shall beinclusive and/or open-ended, shall have the same meaning as the word“comprising” and variants thereof (e.g., “comprise” and “comprises”),and do not exclude additional, un-recited elements or method steps,illustratively.

As used herein, directional and/or arbitrary terms, such as “top,”“bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,”“outer,” “internal,” “external,” “interior,” “exterior,” “proximal,”“distal”, “forward”, “reverse”, and the like can be used solely toindicate relative directions and/or orientations and may not beotherwise intended to limit the scope of the disclosure, including thespecification, invention, and/or claims.

It is also understood that various implementations described herein canbe utilized in combination with any other implementation described ordisclosed, without departing from the scope of the present disclosure.Therefore, products, members, elements, devices, apparatus, systems,methods, processes, compositions, and/or kits according to certainimplementations of the present disclosure can include, incorporate, orotherwise comprise properties, features, components, members, elements,steps, and/or the like described in other implementations (includingsystems, methods, apparatus, and/or the like) disclosed herein withoutdeparting from the scope of the present disclosure. Thus, reference to aspecific feature in relation to one implementation should not beconstrued as being limited to applications only within saidimplementation.

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims. To facilitate understanding, like reference numerals have beenused, where possible, to designate like elements common to the figures.Furthermore, where possible, like numbering of elements have been usedin various figures. Furthermore, alternative configurations of aparticular element may each include separate letters appended to theelement number.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 5%. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; a solution containing one or moremolecules derived from a cell, cellular material, or viral material(e.g. a polypeptide or nucleic acid); a virus; a solution containing oneor more molecules derived from a virus; a parasite; a solutioncontaining one or more molecules derived from a parasite; a bacterium, asolution containing one or more molecules derived from a bacterium, afungus; a solution containing one or more molecules derived from afungus; a plant; a solution containing one more molecules derived from aplant; or a solution containing a nucleic acid, which is assayed asdescribed herein. A sample may also be any body fluid or excretion (forexample, but not limited to, blood, urine, stool, saliva, tears, bile,cerebrospinal fluid, mucous, pus, sweat) that contains cells, cellcomponents, or nucleic acids.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages), or the expanded genetic alphabet described by,for example, Benner U P U.S. Pat. No. 8,354,225, “Amplification ofoligonucleotides containing non-standard nucleobases” and U.S. Pat. No.8,871,469 “Self-avoiding molecular recognition systems in DNA priming”.In particular, nucleic acids can include, without limitation, DNA, RNA,cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandednucleic acid molecule of defined sequence that can base-pair to a secondnucleic acid molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, and the extent of the base-pairing that occurs. Theextent of base-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes, primers, and oligonucleotides may be detectably-labeled, eitherradioactively, fluorescently, or non-radioactively, by methodswell-known to those skilled in the art. dsDNA binding dyes may be usedto detect dsDNA. It is understood that a “primer” is specificallyconfigured to be extended by a polymerase, whereas a “probe” or“oligonucleotide” may or may not be so configured.

By “dsDNA binding dyes” is meant dyes that fluoresce differentially whenbound to double-stranded DNA than when bound to single-stranded DNA orfree in solution, usually by fluorescing more strongly. While referenceis made to dsDNA binding dyes, it is understood that any suitable dyemay be used herein, with some non-limiting illustrative dyes describedin U.S. Pat. No. 7,387,887, herein incorporated by reference. Othersignal producing substances also may be used for detecting nucleic acidamplification and melting, illustratively enzymes, antibodies, etc., asare known in the art.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions configured to allowfor identification of target nucleic acid sequences. Such conditionstypically occur at about Tm minus 5° C. (5° below the Tm of the probe).Functionally, high stringency conditions are used to identify nucleicacid sequences having at least 80% sequence identity.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependent amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. For amplification methodswithout discrete cycles, reaction time may be used where measurementsare made in cycles or Cp, and additional reaction time may be addedwhere additional PCR cycles are added in the embodiments describedherein. It is understood that protocols may need to be adjustedaccordingly.

In some embodiments, PCR includes any suitable PCR method. For example,PCR can include multiplex PCR in which the polymerase chain reaction isused to amplify several different nucleic acid sequences simultaneously.Multiplex PCR can use multiple primer sets and can amplify severaldifferent nucleic acid sequences at the same time. In some cases,multiplex PCR can employ multiple primer sets within a single PCRreaction to produce amplicons of different nucleic acid sequence thatare specific to different nucleic acid target sequences. Multiplex PCRcan have the helpful feature of generating amplicons of differentnucleic acid target sequences with a single PCR reaction instead ofmultiple individual PCR reactions.

As used herein, “sequencing” means identifying the sequence of a nucleicacid, including identifying the sequence of one or both strands of thenucleic acid, to determine a sequential order of the individual bases.“Next-generation sequencing” is sequencing performed on a plurality ofsimultaneous parallel reactions.

The following nomenclature for oligonucleotides is used herein:

-   -   U=Universal primer—common sequence shared by all primers in a        particular reaction    -   S=Specific primer—unique to a given target assay Subscripts:        -   o=outer—primer used in the first-stage amplification to            generate the outer amplicon        -   i=inner—a nested primer used in the second-stage            amplification reaction. This primer can partially overlap            the outer primer that was used to generate the outer            amplicon, but lies partially or wholly inside the outer            amplicon        -   ii=inner-inner—a primer that nests inside of the nested            inner primer. This primer can partially overlap this inner            primer or sit wholly inside the inner primer        -   F=Forward and R=Reverse—are the two primers that define a            PCR amplicon. It is understood that the orientation is            arbitrary        -   x=index of a particular specific oligonucleotide in the            multiplex (1≤x≤n)            For example: U_(R)S_(IR4) is a universal reverse primer on            the 5′ end of Specific inner reverse for amplicon number 4.

While various examples herein reference human targets and humanpathogens, these examples are illustrative only. Methods, kits, anddevices described herein may be used to detect and sequence a widevariety of nucleic acid sequences from a wide variety of samples,including, but not limited to human samples, animal sample, veterinarysamples, plant samples, algae samples, food samples, industrial samples,viral samples, fungus samples, bacteria samples, parasite samples, andenvironmental samples. The methods, kits, and devices described hereinmay be used to detect and sequence a wide variety of nucleic acidsequences from a wide variety of applications including, but not limitedto, detecting antibiotic resistance, detecting viral genes, forensics,tissue typing, organ transplantation, drug metabolism, bioterrorism,food safety, environmental safety, agriculture, and ecology.

Various embodiments disclosed herein use a self-contained nucleic acidanalysis pouch to assay a sample for the presence of and/oridentification of various biological substances, illustratively antigensand nucleic acid sequences, illustratively in a single closed system.Such systems, including pouches and instruments for use with thepouches, are disclosed in more detail in U.S. Pat. Nos. 8,394,608; and8,895,295; and U.S. Patent Application No. 2014-0283945, hereinincorporated by reference. However, it is understood that such pouchesare illustrative only, and the PCR reactions discussed herein may beperformed in any of a variety of open or closed system sample vessels asare known in the art, including 96-well plates, plates of otherconfigurations, arrays, carousels, and the like, using a variety ofamplification systems, as are known in the art. While the terms “samplewell”, “amplification well”, “amplification container”, or the like areused herein, these terms are meant to encompass wells, tubes, DNA chips,and various other reaction containers, as are used in theseamplification systems. In one embodiment, the pouch is used to assay formultiple target nucleic acids. The pouch may include one or moreblisters used as sample wells, illustratively in a closed system.Illustratively, various steps may be performed in the optionallydisposable pouch, including nucleic acid preparation, primary largevolume multiplex PCR, dilution of primary amplification product, andsecondary PCR, culminating with optional real-time detection orpost-amplification analysis such as melting-curve analysis and nucleicacid sequencing. Further, it is understood that while the various stepsmay be performed in pouches of the present invention, one or more of thesteps may be omitted for certain uses, and the pouch configuration maybe altered accordingly.

FIG. 1 shows an illustrative pouch 510 that may be used in variousembodiments, or may be reconfigured for various embodiments. Pouch 510is similar to FIG. 15 of U.S. Pat. No. 8,895,295, with like itemsnumbered the same. Fitment 590 is provided with entry channels 515 athrough 515 l, which also serve as reagent reservoirs or wastereservoirs. Illustratively, reagents may be freeze dried in fitment 590and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566,with their respective channels 538, 543, 552, 553, 562, and 565 aresimilar to blisters of the same number of FIG. 15 of U.S. Pat. No.8,895,295. Second-stage reaction zone 580 of FIG. 1 is similar to thatof U.S. Pat. No. 8,895,295, but the second-stage wells 582 of highdensity array 581 are arranged in a somewhat different pattern. The morecircular pattern of high density array 581 of FIG. 1 eliminates wells incorners and may result in more uniform filling of second-stage wells582. As shown, the high density array 581 is provided with 102second-stage wells 582. Pouch 510 is suitable for use in the FilmArrayinstrument (BioFire Diagnostics, Salt Lake City, Utah). However, it isunderstood that the pouch embodiment is illustrative only.

Pouch 510 may be used in a manner similar to that described in U.S. Pat.No. 8,895,295. In one illustrative embodiment, a 300 μl mixturecomprising the sample to be tested (100 μl) and lysis buffer (200 μl) isinjected into injection port (not shown) in fitment 590 near entrychannel 515 a, and the sample mixture is drawn into entry channel 515 a.Water is also injected into a second injection port (not shown) of thefitment 590 adjacent entry channel 515 l, and is distributed via achannel (not shown) provided in fitment 590, thereby hydrating up toeleven different reagents, each of which were previously provided in dryform at entry channels 515 b through 515 l via. These reagentsillustratively may include freeze-dried PCR reagents, DNA extractionreagents, wash solutions, immunoassay reagents, or other chemicalentities. Illustratively, the reagents are for nucleic acid extraction,first-stage multiplex PCR, dilution of the multiplex reaction, andpreparation of second-stage PCR reagents, as well as control reactions.In the embodiment shown in FIG. 1, all that need be injected is thesample solution in one injection port and water in the other injectionport. After injection, the two injection ports may be sealed. For moreinformation on various configurations of pouch 510 and fitment 590, seeU.S. Pat. No. 8,895,295, already incorporated by reference.

After injection, the sample is moved from injection channel 515 a tolysis blister 522 via channel 514. Lysis blister 522 is provided withceramic beads 534 and is configured for vortexing via impaction usingrotating blades or paddles provided within the FilmArray instrument.Once the cells have been adequately lysed, the sample is moved throughchannel 538, blister 544, and channel 543, to blister 546, where thesample is mixed with nucleic acid-binding magnetic beads, such assilica-coated magnetic beads 533. The mixture is allowed to incubate foran appropriate length of time, illustratively approximately 10 secondsto 10 minutes. A retractable magnet located within the FilmArrayinstrument adjacent blister 546 captures the magnetic beads from thesolution, forming a pellet against the interior surface of blister 546.The liquid is then moved out of blister 546 and back through blister 544and into blister 522, which is now used as a waste receptacle. One ormore wash buffers from one or more of injection channels 515 c to 515 eare provided via blister 544 and channel 543 to blister 546. Optionally,the magnet is retracted and the magnetic beads are washed by moving thebeads back and forth from blisters 544 and 546 via channel 543. Once themagnetic beads are washed, the magnetic beads are recaptured in blister546 by activation of the magnet, and the wash solution is then moved toblister 522. This process may be repeated as necessary to wash the lysisbuffer and sample debris from the nucleic acid-binding magnetic beads.

After washing, elution buffer stored at injection channel 515 f is movedto blister 548, and the magnet is retracted. The solution is cycledbetween blisters 546 and 548 via channel 552, breaking up the pellet ofmagnetic beads in blister 546 and allowing the captured nucleic acids todissociate from the beads and be released into solution. The magnet isonce again activated, capturing the magnetic beads in blister 546, andthe eluted nucleic acid solution is moved into blister 548.

First-stage PCR master mix from injection channel 515 g is mixed withthe nucleic acid sample in blister 548. Optionally, the mixture is mixedby forcing the mixture between 548 and 564 via channel 553. Afterseveral cycles of mixing, the solution is contained in blister 564,where a pellet of first-stage PCR primers is provided, at least one setof primers for each target nucleic acid sequence, and first-stagemultiplex PCR is performed. If RNA targets are present, an RT (reversetranscription) step may be performed prior to or simultaneously with thefirst-stage multiplex PCR. First-stage multiplex PCR temperature cyclingin the FilmArray instrument is illustratively performed for 15-30cycles, although other levels of amplification may be desirable,depending on the requirements of the specific application. Thefirst-stage PCR master mix may be any of various master mixes, as areknown in the art. In one illustrative example, the first-stage PCRmaster mix may be any of the chemistries disclosed in U.S. Ser. No.14/403,369 and WO2013/177429, already incorporated by reference, for usewith PCR protocols taking 20 seconds or less per cycle.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted, illustratively by forcing most of the sampleback into blister 548, leaving only a small amount in blister 564, andadding second-stage PCR master mix from injection channel 515 i.Alternatively, a dilution buffer from 515 i may be moved to blister 566then mixed with the amplified sample in blister 564 by moving the fluidsback and forth between blisters 564 and 566. If desired, dilution may berepeated several times, using dilution buffer from injection channels515 j and 515 k, or injection channel 515 k may be reserved forsequencing, as discussed below, and then adding second-stage PCR mastermix from injection channel 515 h to some or all of the diluted amplifiedsample. It is understood that the level of dilution may be adjusted byaltering the number of dilution steps or by altering the percentage ofthe sample discarded prior to mixing with the dilution buffer orsecond-stage PCR master mix comprising components for amplification,illustratively a polymerase, dNTPs, and a suitable buffer, althoughother components may be suitable, particularly for non-PCR amplificationmethods. If desired, this mixture of the sample and second-stage PCRmaster mix may be pre-heated in blister 564 prior to movement tosecond-stage wells 582 for second-stage amplification. Such preheatingmay obviate the need for a hot-start component (antibody, chemical, orotherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 102 second-stage wells 582 is individuallypre-loaded with a specific PCR primer pair. If desired, second-stage PCRmaster mix may lack other reaction components, and these components maybe pre-loaded in the second-stage wells 582 as well. Each primer pairmay be similar to or identical to a first-stage PCR primer pair or maybe nested within the first-stage primer pair. It is understood thatnested primers are primers that hybridize to the first-stage amplicon ina position that is internal to the hybridization location of thefirst-stage primers. A nested primer may partially overlap thefirst-stage primer but the 3′-end is located internally to the 3′-end ofthe first-stage primer. Movement of the sample from blister 564 to thesecond-stage wells 582 completes the PCR reaction mixture. Once highdensity array 581 is filled, the individual second-stage reactions aresealed in their respective second-stage blisters by any number of means,as is known in the art. Illustrative ways of filling and sealing thehigh density array 581 without cross-contamination are discussed in U.S.Pat. No. 8,895,295, already incorporated by reference. Illustratively,the various reactions in wells 582 of high density array 581 aresimultaneously thermal cycled to carry out the second-stage PCRreaction, illustratively with one or more peltier devices, althoughother means for thermal cycling are known in the art.

In certain embodiments, second-stage PCR master mix contains the dsDNAbinding dye LCGreen® Plus to generate a signal indicative ofamplification. However, it is understood that this dye is illustrativeonly, and that other signals may be used, including other dsDNA bindingdyes, and probes that are labeled fluorescently, radioactively,chemiluminescently, enzymatically, or the like, as are known in the art.Alternatively, wells 582 of array 581 may be provided without a signal,with results reported through subsequent sequencing.

The illustrative FilmArray instrument can be programmed to make positiveor negative calls for each second-stage reaction based on a post-PCRmelt. A positive call can indicate a successful second-stage PCRreaction for a corresponding well 582. A positive call can also indicatethat the quality of the amplicon generated in the second-stage PCRreaction is sufficient to permit sequencing. A positive call can alsoindicate that the second-stage PCR reaction has produced an ampliconthat is a substantially homogeneous molecular species. A negative callcan indicate an unsuccessful second-stage PCR reaction for acorresponding well 582. A negative call can also indicate that thequality of the amplicon generated in the second-stage PCR reaction isnot of sufficient to permit sequencing. A negative call can alsoindicate that the second-stage PCR reaction has not produced an ampliconthat is a substantially homogeneous molecular species. In oneembodiment, the melt curve must produce a melt peak (e.g., firstderivative maximum or negative first derivative maximum) within apre-defined temperature range, for the call to be positive. It isunderstood that this method of calling each second-stage reaction isillustrative only, and that calls could be made using real-timeamplification data or by other means, as are known in the art, or callsmay be deferred until subsequent sequencing is performed.

Illustratively, if the instrument is configured to make positive ornegative calls, the instrument may be programmed to earmark only thosewells that produce a positive result for subsequent sequencing. In someembodiments, there may be a one-to-one unity between positive calls andthe wells earmarked for sequencing. In other embodiments, the wellsearmarked for sequencing may not have one-to-one unity with the wellsthat produce a positive call. For example, the second-stage 580 of theFilmArray pouch 510 may be spotted with replicate wells, illustrativelyduplicate or triplicate wells. If one or more of those wells come uppositive, it may be desirable for all replicates of that target sequenceto be earmarked for sequencing. In other embodiments, it may bedesirable to earmark only one well for each replicate for sequencing. Inyet other embodiments, wells that are used as controls may or may not beearmarked for subsequent sequencing. In yet other embodiments, all wellsmay be sequenced, with or without positive or negative amplificationcalls.

While the FilmArray is useful in providing positive or negative resultsbased on PCR from a crude biological sample, methods of going from acrude biological sample to the sequence of specific RNA and DNA targetsalso would be useful. In one illustrative embodiment, the procedure maybe automated, may take less than 8 hours, more illustratively less than5 hours, and more illustratively take 3 hours or less, and may provide25 or more bases of sequence, illustratively up to 200 bases of sequencefor multiple amplicons, and illustratively up 1000 amplicons.Illustratively, crude sample-to-sequence processes will occur in aclosed system so as to minimize the chance of amplicon contamination.

The second-stage 580 of the FilmArray pouch 510 is used to generateamplicons for the desired target. Because of the two-step PCR,essentially homogeneous amplicons are generated in each of thesecond-stage wells 582. Each batch of amplicons in its respective well582 may be independently sequenced by sequencing methods known in theart. For example, each batch of amplicons can be sequenced by any one ofseveral techniques known in the art for Next Generation Sequencing(NGS), such as Ion Torrent (Life Technologies), 454 (Roche), SOLiD(ABI), HiSeq or MiSeq (Illumina), and Pacific Biosciences.

In many successful FilmArray pouch runs, a positive signal in a well 582in the second-stage array 581 represents a substantially pure singlespecies of amplicon. This single species of amplicon is not necessarilya molecular clone. For example, digital PCR, emulsion PCR, or molecularcloning into a plasmid are all methods that generate spatially separatedmolecular clones of an original nucleic acid molecule. In contrast, thesecond-stage amplicon originates from many identical or closely relatedtemplate molecules. However, because of the first-stage enrichment anddilution, the end result of the second-stage PCR is predominantly asingle species of amplicon, referred to herein as a “single molecularspecies”. It is understood that, as used herein, a single molecularspecies will not necessarily be a molecular clone, but will besufficiently one predominant species to enable sequencing.

In some embodiments, melting curves of the amplicons generated in thesecond-stage PCR indicate that these amplicons are essentiallyhomogeneous. That the melting curve analysis is performed on theFilmArray instrument with the non-specific double-stranded DNA bindingdye LCGreen® Plus and results in single peaks and indicates that thesecond-stage PCR product is dominated by a single intended amplicon anddoes not include a significant amount of primer-dimer species ornon-specific amplicons, demonstrating that the two step nested multiplexamplification generates single molecular species.

In some embodiments, a target nucleic acid sequence is present in a verylow copy number in a sample. The target nucleic acid can comprise a verysmall portion of the overall sample and/or there can be other nucleicacids present. For example, in PCR-based pathogen detectionapplications, the pathogens of interest may be present at very low copynumber in a sample, and the sample may be contaminated with a largeexcess of other nucleic acids, illustratively human nucleic acid (e.g.,blood) or other microbial genomes (e.g., stool) and may result in acomplex nucleic acid mixture. In conventional single-stage PCR detectionsystems, the single-stage PCR amplification from this complex nucleicacid mixture often results in multiple non-specific products in additionto the desired specific amplicon. Therefore conventional single-stagePCR detection systems often require a second detection mechanism or“filter”, in addition to the basic single-stage PCR to isolate thecorrect amplicon and/or to confirm the identification of the correctamplicon. This filter can include a size exclusion step (e.g., agaroseor acrylamide gel) to identify the target amplicon, a probe baseddetection method (e.g., using a hydrolysis probe or two hybridizationprobes) or sequence capture method using sequence capture to a surface(GenMark Diagnostics, Inc., Carlsbad, Calif.) or to a bead (LuminexCorporation, Austin, Tex.) that can be sorted.

In some embodiments, because two-step PCR generates amplicons that areessentially homogeneous, the second detection mechanism or “filter” canbe omitted and the amplicons generated in the second-stage PCR can bedirectly sequenced. Thus the amplicons generated by the second-stage PCRcan be directly sequenced by any means known in the art.

Illustratively, it should be possible to sequence the population bytethering a portion of the amplicon to the well, illustratively bycovalently linking a second-stage primer or the amplicon to the bottomof the well, and then using a next generation sequencing method that maybe based on the NGS technologies of Ion Torrent, 454, Illumina or ABI(SoLiD), or other sequencing methods, as are known in the art. Unlikeother systems, this subsequent sequencing may be accomplishedautomatically, within one or more of the second-stage wells.

Example 1

In this example, one strand of the amplicon is tethered to the bottom ofthe well (FIG. 2A) prior to sequencing. In one illustrative example, oneway to provide tethered amplicon would be to synthesize one of theoligonucleotides (illustratively the forward primer) in two forms, onewith the normal 5′ OH and the other with a 5′ extension that has achemical moiety provided on the 5′ end that allows it to be cross linkedto the well surface. There are a variety of chemistries that are knownin the art for this purpose. This would provide some of the forwardprimer tethered to the reaction well and some of the forward primer freein solution. It is understood that the forward primer free in solutionis provided to improve the kinetics of the amplification reaction. Oneillustrative way to load the second-stage array 581 would be to spot thearray twice, illustratively using a device such as that described in WO2013/158740 and U.S. Ser. No. 14/395,002, herein incorporated byreference. Illustratively, the first time array 581 is spotted, thechemically modified primers 24 are added into each well 582 of array581. The chemically modified primers 24 are then cross-linked to thewell 582 and subsequently the coupling reaction may be quenched toprevent further crosslinking of molecules to the array. The array 581may be dried and then spotted again with reverse primers 32, andoptionally other PCR components. Optionally, the unmodified forwardprimers 26 may be spotted to provide increased primer concentrations toimprove the kinetics of the PCR amplification. However, this method forspotting array 581 is illustrative only, and it is understood that thechemically modified primers 24 (as shown in FIG. 2A), unmodified forwardprimers 26, and reverse primers 32 may be spotted at one time, or in anyorder. In other embodiments, the chemically modified primer 24 may bespotted together with or prior to spotting the reverse primer 32, andthe unmodified forward primer 26 may be omitted. Illustratively, thereverse primer 32 may have a 5′ extension sequence 20 that is the samefor all reverse primers in the array 581. This “universal primer” isprovided to be used later during the sequencing reaction. In theillustrative embodiment with some forward primer provided free insolution, most of the PCR reaction will occur in solution but a fractionof the amplicons 28 will be in the form of a tethered amplicon 29 thatis tethered to the well 582 in array 581 (see FIG. 2A).

Many of the examples herein describe tethering one or more primers tosample wells or spots on an array. However, it is understood that whentethering is discussed, this may include tethering to any solid surface,including beads, that can be moved to sequencing locations.

Example 2

In this example, an illustrative example of a geometry of thesecond-stage PCR array 580 as a sequencing chip is described. FIG. 3shows a partially exploded cross-sectional view of illustrative array581. In this illustrative embodiment, a silicon layer 589 is built ontothe bottom of array 581, illustratively replacing second layer 587 (notshown), as discussed in U.S. Pat. No. 8,895,295, and forming a bottomsurface in each of wells 582.

As mentioned above, illustrative ways of filling and sealing the highdensity array 581 without cross-contamination are discussed in U.S. Pat.No. 8,895,295, already incorporated by reference. In one illustrativeembodiment, shown in FIG. 3, a pierced layer 585 is provided, similar topierced layer 585 of U.S. Pat. No. 8,895,295. Pierced layer 585 allowsfluid to pass into each well 582 in the presence of a force, but thepiercings are small enough to substantially prevent fluid from passingin the absence of the force. However, it is understood that a chemicalmechanism for keeping the primers in place during rehydration may be asor more desirable than the pierced layer in the present embodiment, toprovide less interference in the subsequent sequencing step. In oneembodiment employing a chemical mechanism, the “solution phase” forwardprimers 26 and reverse primers 32 may be combined with a solution ofmolten low melt agarose, for example UltraPure™ Low Melting PointAgarose (Life Technologies), before spotting so that they do not washout of the array when it is first flooded, although other materials forthe chemical mechanism are known in the art, some of which are discussedin U.S. Pat. No. 8,895,295. After flooding second-stage array 581, thewells 582 in array 581 are sealed shut by inflating a bladder in theinstrument, discussed in U.S. Pat. No. 8,895,295, and pushing againstfilm 518, which is the same or similar to layer 518, as described inU.S. Pat. No. 8,895,295. In embodiments using low melt agarose, once thewells 582 are sealed, heating of the array releases forward primer 26and reverse primer 22 into solution. Array 581 may be made largely ofsilicon, or may be provided with silicon layer 589, with the remainderof materials similar to array 581, as discussed in U.S. Pat. No.8,895,295, or may be made of other materials and is configured such thatliquid flowing across array 581 is able to enter the wells 582 andintroduce new chemicals or wash out previous chemicals. It may bedesirable to provide array 581 with a depth of from about 50 μm to about500 μm, but other sizes may be appropriate, depending on sequence methodand configuration.

Referring to FIG. 1, second-stage reaction zone 580 is provided with twoports, channel 565 and channel 567, each of which may be sealed bypressure or other means. Channel 565 is provided for receiving fluidfrom blister 566, which is shown connected to multiple entry channels.Injection channel 515 k may contain materials for sequencing, or mayhave a valve or sealable port, allowing connection to an external sourcefor the materials needed for sequencing. Similarly injection channel 515l may receive the waste materials from sequencing or may be providedwith a valve or sealable port for connection to a separate wastereceptacle. Thus, in this illustrative embodiment, liquid flows frominjection channel 515 k, through channel 565, across all wells 582 ofarray 581 and outlet channel 567, to waste reservoir 515 l. It isunderstood that, as fluid is moved into second-stage amplification zone580, outlet channel may remain sealed. This will form a bubble of fluidover array 581, and agitation can create a homogenized fluid above thearray. Synchronized opening and closing of channels 565 and 567 cancontrol flow of fluid across array 581.

Subsequent to amplification of first stage amplicon 21 (FIG. 2A (i) and(ii)), sequencing illustratively may be performed by the followingsteps:

-   -   1) Denaturing the tethered amplicon 29, illustratively by        heating, and washing the unbound strand 30 from the array (FIG.        2A (ii) and FIG. 2A (iii)). It is understood that this will also        wash out all unbound amplicon 28.    -   2) Annealing sequencing primer 32 to universal tail 20 and        providing a DNA polymerase 34 to form an initiation complex.        Washing out unbound sequencing primer and unbound DNA polymerase        (FIG. 2B (iv) and (v))    -   3) Sequencing. Sequencing may be performed by synthesis as the        DNA polymerase moves along the bound strand 29 and amplifies the        sequence of the bound strand 29. Each of the nucleotides A, T,        C, and G are individually provided and incorporation of the        individual dNTPs is measured, thereby revealing the nucleotide        sequence of bound strand 29. The incorporation of individual        dNTPs can be measured by methods know in the art (e.g., proton        generation (as with Ion Torrent), fluorescence or        chemiluminescence (as with Roche 454), or any other signal as is        appropriate for the sequencing method). Array 581 is washed, and        this is repeated until the sequence has been generated (FIG.        2B (vi) and (vii)).

In an alternative embodiment, PCR may be performed in pouch 510 withreal-time curves and/or a melt, and then each well 582 is sealed andsecond-stage amplification zone 580 of pouch 510 is removed to a secondinstrument for sequencing. Tethering the forward primer, as discussedabove, may be one way to prevent well-to-well contamination, althoughother ways may be possible, illustratively by sealing wells 582. Thus,tethering may be optional, depending on the sequencing method used.

It is understood that other sequencing methods may be used in such asystem. One such illustrative method would not require linking theamplicon to the well and would rather use a single molecule sequencingmethod (Oxford Nanopore, see Wolna A H, et al. Electrical CurrentSignatures of DNA Base Modifications in Single Molecules Immobilized inthe α-Hemolysin Ion Channel. Isr J Chem. 2013 Jun. 1; 53(6-7):417-430.PubMed PMID:24052667; PubMed Central PMCID: PMC3773884). In such analternate embodiment, the array well 582 may be connected to orintegrated with a chamber with a single channel and the population ofsecond-stage amplicons in the well 582 is read one base at a time as theamplicon is translocated through the channel. This connection to thechamber could be built into pouch 510 or it could be made after thesecond-stage PCR run is completed (as long as the amplicons are keptsegregated into their individual wells). Illustratively, afteramplification, one side of the array 581 is sealed onto a layer of filmthat has a single protein nanopore channel disposed over each well. Onthe other side of the channel is additional fluid so that an electriccurrent can be applied between the well 582 and the additional fluid andthrough the protein nanopore channel Sequencing can then be performed onthe amplicons as they migrate through the protein nanopore channels asper Oxford Nanopore, or by similar methods. Illustratively, OxfordNanopore uses the protein α-hemolysin as the protein nanopore channel.The protein nanopore is disposed in a lipid bilayer and has asufficiently sized opening to translocate a single stranded DNA moleculethrough the opening. As the single-stranded DNA molecule traverses thenanopore, protons and other ions that otherwise flow across the nanoporeare impeded and consequently current flow across the nanopore isaltered. In the Oxford Nanopore system, the identity (A, T, G, or C) ofindividual bases of the single-stranded DNA molecule can be individuallydiscerned by this alteration in current flow. Thus, by measuring changesin electrical potential caused by current alterations corresponding toindividual base pairs, the sequence of the individual nucleotides withinthe single-stranded DNA molecule can be ascertained. (See Maglia,Giovanni, et al. “Analysis of single nucleic acid molecules with proteinnanopores.” Methods in enzymology 475 (2010): 591-623 and Clarke, James,et al. “Continuous base identification for single-molecule nanopore DNAsequencing.” Nature Nanotechnology 4.4 (2009): 265-270.) However, it isunderstood that the Oxford Nanopore system is illustrative and thatother sequencing configurations are contemplated. In another version,similar to the method developed by Pacific Biosciences of Menlo Park,Calif., a sequencing well comprises zero-mode waveguides on a bottomsurface that create a small light detection volume. Each zero-modewaveguide is illuminated from below by an excitation beam and thezero-mode waveguide prevents the wavelength of light from efficientlypassing through the waveguide thereby allowing attenuated light from theexcitation beam to penetrate the lower 20-30 nm of each zero-modewaveguide. A complex of the amplicon and a DNA polymerase complex isthen tethered to the bottom of the zero-mode waveguide. Phospho-linkednucleotides are then added to the well. Each of the four nucleotides canbe labelled with a different colored fluorophore. As the DNA polymerasecomplex amplifies the amplicon, the individual phospho-linkednucleotides are incorporated into the nascent strand and the fluorescentcolor of each individual phosphor-linked nucleotide can be detected insequential order to determine the nucleotide sequence of the amplicon.Since there are multiple polymerases per original well, theincorporation of nucleotides into each strand held by each polymeraseshould be synchronized for best results.

In one illustrative example for demonstrating that a single molecularspecies results from two-step nested multiplex PCR, second-stageamplicons from a FilmArray run have been sequenced. As part of aninvestigation of false positive test results in the FilmArray BCID Panel(BioFire Diagnostics), a mixture of organisms including Candidaglabrata, Neisseria meningitidis, Escherichia coli, and Streptococcusmitis, was tested using a commercial FilmArray BCID Panel andexperimental software. A weak false positive result with very late Cps,indicative of poor amplification, was detected in the Candida kruseisecond-stage wells.

Amplicon from each of these positive second-stage wells was extractedfrom the pouches by piercing of each of the wells on the second-stagearray with an insulin syringe and withdrawing the solution containingthe amplicon. The amplicon was then amplified in a CFX instrument(Bio-Rad) for an additional 20 cycles using the same Candida kruseiinner primers as used in the second-stage of the BCID Panel. Thisfurther amplified material was then sequenced by Sanger sequencing on anABI instrument. Readable sequence could be obtained using both theforward and reverse primers with Phred quality scores above 20 for 51nucleotides (forward primer) and 75 nucleotides (reverse primer). Table1 shows the results of sequence comparison using the BLAST algorithm ofthe contiguous sequence (made from the overlapping regions of theforward and reverse sequences) against the GenBank database (NationalCenter for Biotechnology Information).

TABLE 1 Max Total Query Description Score Score cover E value IdentAccession Streptococcus mitis 194 194 99% 3e−46 97% CP014326.1 strainSVGS_061, complete genome Streptococcus 194 194 99% 3e−46 97% CP003357.2pneumoniae ST556, complete genome Streptococcus 194 194 99% 3e−46 97%LN847353.1 pneumoniae genome assembly S_pneumo_A66_v1, chromosome: 1Streptococcus 194 194 99% 3e−46 97% LN831051.1 pneumoniae genomeassembly NCTC7465, chromosome: 1 Streptococcus 194 194 99% 3e−46 97%CP007593.1 pneumoniae strain NT_110_58, complete genome Streptococcus194 194 99% 3e−46 97% CP006844.1 pneumoniae A026 genome Streptococcus194 194 99% 3e−46 97% PQ312041.2 pneumoniae SPN994038 draft genome

These data show that the C. krusei assay in the second-stage of the BCIDPanel is specifically amplifying a region of the Streptococcus mitisgenome. This demonstrates that good sequence data can be obtained fromthe two-step nested multiplex PCR in the BCID Panel even when the PCRthat produced the sequence had a very late Cp, indicating that theamplicon was not abundant.

Example 3

In this example, illustrative embodiments of nucleotide sequencing in atwo-step PCR system are described. Referring now to FIG. 4, FIG. 4 issimilar to FIG. 1, with like reference numerals indicating similarcomponents. In this embodiment, array 580 with wells 582 are replaced bya microarray 680 with spots 682 (often referred to as features). Spots682 are areas of the array that have at least one oligonucleotidedeposited thereon. While having one homogenous group of oligonucleotidesspotted per spot 682 is used illustratively herein, it is understoodthat each spot 682 may have both members of a primer pair, or may have aset of nested oligonucleotides spotted thereon, and other configurationsare possible. Thus, it is understood that each spot 682 has one set ofoligonucleotides spotted thereon, where each set is one or moreoligonucleotide species, and the set is specific for a target.

In one illustrative embodiment, one oligonucleotide species is spottedin each spot 682, which is used to extend one end of an amplicon, and aplurality of spots 682 each has a different oligonucleotide speciesspotted thereon. It is understood that each spot 682 of array 680 mayhave a different species, or there may be replicate (illustrativelyduplicate or triplicate) spots, or any combination thereof.

In another illustrative embodiment that will be discussed in more detailbelow, two members of a primer pair are spotted at the same spot 682 onthe array, allowing for bridge PCR at that spot, so that the whole spot682 becomes covered with amplicon that is anchored by one or the otherend. Prior art next generation sequencers that use bridge PCR randomlydeposits primers across the whole flow cell, permitting amplification ofany single member of the library that has been deposited on the array.By spotting both members of a primer pair in a single spot 682, asequence will be obtained from that spot 682 only if the target sequenceis present. Thus, obtaining a sequence from that spot not only providesthe sequence, but can also provide a positive call for that target.

Illustratively, the substrate material for array 680 may be glass. Manymicroarrays are made on a glass surface, illustratively because: (1) thechemistry for coupling oligonucleotides to glass at high loading densityis well understood; (2) the flatness and lack of stretching means thatarray printers can place small spots very close to each other; and (3)glass has relatively low auto-fluorescence, so that the signal-to-noiseratio for a fluorescent dye on the array is easier to detect.Integration of a glass array into the pouch 610 may require putting ahard frame around array 680 or around pouch 610, to reduce breakage of aglass array 680.

However, it is understood that other materials may be used for array680. Methods are known in the art for attaching oligonucleotides toplastic. In some embodiments, a plastic array can have one or more ofthe following: (1) plastic bonds to the other plastic components easilyand can be more readily integrated into pouch 610, (2) a flexibleplastic may be used, so that array 681 is less breakable if pouch 610 isstressed. A plastic microarray may work well in pouch 610 because (a)one can make relatively large spots so the signal will relatively largeand the accuracy of spotting is not critical (256 spots on a 1 square cmarray area=square spots of 0.39 mm²). A spot of this size is quite largeby microarray standards. By comparison the commercial FilmArray wellsare circles of 1.98 mm² area (the wells are 1.59 mm in diameter). Largerspots will generate larger signals which means that the camera forimaging fluorescent nucleotide incorporation will need fewer sensingelements or the silicon chip used to sense pH changes will require fewertransistors, which will make both devices less expensive to manufacture,and (b) plastics are known that have low auto-fluorescence and can stillbind oligonucleotides at a relatively high density. It is understoodthat an array of 512 spots or fewer, or 256 spots or fewer, or anynumber of spots are contemplated.

It is understood that the illustrative arrays described herein can havethe oligonucleotides tethered at the 5′ end to the well 682. In thatsense they are different from many other arrays known in the art, e.g.Affymetrix arrays, that have the oligonucleotide attached by its 3′ endto the substrate (e.g., the well). Accordingly, Affymetrix arrays canonly be used for hybridization, not for extension as a primer in anamplification reaction as in the illustrative arrays.

A DNA polymerase with a 3′ to 5′ exonuclease activity (for example T4DNA polymerase, T7 DNA polymerase or Vent DNA polymerase), in additionto acting as a DNA polymerase can perform the following additionalactivities (in the presence of dNTPs): (1) remove bases from a 3′overhang of a double-stranded DNA and (2) degrade single-stranded DNA tonucleotides. (See New England BioLabs, New England. “New England BioLabscatalog and technical reference.” Beverly, Mass.: New England Biolabs(2014) and BEBENEK, K. et al. (1989) Nucl. Acids Res. 17, 5408, hereinincorporated by reference.) In some embodiments, these 3′ to 5′exonuclease DNA polymerases with these additional activities are usefulin two-step nested multiplex PCR. In certain commercial embodiments, atthe end of first-stage PCR, the reaction mixture is be diluted,illustratively 100 to 250-fold, prior to the nested second-stagereactions, and new inner nested primers are used (primers that arenested within the first-stage primers). This is done to prevent thefirst-stage PCR primers from continuing to amplify any primer dimers,which avoids a non-specific rising baseline in the amplification curveand a large, broad, melt peak in second-stage PCR. An illustrativetreatment a DNA polymerase with 3′ to 5′ exonuclease activity (e.g., anexo+ DNA polymerase) in the presence of dNTPs will degrade thefirst-stage primers that have not been incorporated into double-strandedamplicon. The correct amplicons, non-specific amplicons and primerdimers are all double-stranded, so they should remain intact. Withoutthe first-stage PCR primers, the primer dimers present from first-stagePCR should not amplify in second-stage PCR, thus enabling a smallerdilution (or no dilution) between first-stage PCR and second-stage PCR,which means that fewer cycles will be needed to get a second-stageamplicon to appear above background (have an earlier Cp in the secondstage reaction).

In addition, the exo+ DNA polymerase treatment will degrade segments ofhigh complexity genomes (such as the human genomic DNA, human mRNAconverted to cDNA (when an RT-PCR step is used), as well asnon-amplified pathogens and contamination that may be present) that arein low copy number. This may happen because standard PCR cyclingconditions (in particular the annealing step) may be fast enough toprevent low copy high complexity DNA from reannealing, and thus will bedegraded by the exo+ activity, although some sequences that are presentin higher copy numbers may reanneal. An advantage of using exonucleaseat this step is that it reduces the sequence complexity of the DNA thatenters the second-stage PCR reaction. This can result in lessnon-specific amplification in second-stage PCR that could be detected bya double-stranded DNA binding dye, such as LCGreen. Thus, it can bepossible to add additional cycles to second-stage PCR with less signalinterference from mis-amplification.

In one illustrative example, in pouch 610, two successive dilutions ofabout 10 to 15-fold each, equaling 100 to 225-fold total dilution, aremade using components from injection channels 616 h, 615 i, and/or 615j. As an alternative to performing this level of dilution, in oneillustrative embodiment, a small amount of the exo+ polymerase can beadded directly to the first-stage PCR at the end of cycling,illustratively from injection channel 615 h. This may require a longerdigestion because of the large number of primers present in thehighly-multiplexed first-stage PCR. It is understood that most of theseprimers are still single stranded, as only a few targets usually amplifyin any single run. However, any advantage in reduced speed insecond-stage PCR may be lost by the time it takes to perform this largedigestion. Thus, it may be desirable to perform a single dilution,illustratively 10 to 15 fold, prior to or simultaneously with the exo+digestion. Illustratively, if the exo+ enzyme is T4 or T7, the mixturemay be incubated at 37 to 42° C. respectively for a time sufficient toallow the first-stage PCR primers to be degraded. At the end of thisstep, the mixture may be heated to 80 to 90° C. for a time long enoughto inactivate the exo+ enzyme. This mixture can then be flooded acrossthe second-stage array 681.

In some embodiments, Vent DNA polymerase with 3′ to 5′ exonuclease isused and steps must be taken to prevent inactivation of the second-stagePCR primers by the Vent Polymerase. Vent Polymerase is not thermolabileso it cannot be inactivated by heat before carrying out the second-stagePCR amplification. To prevent inactivation of the second-stage PCRprimers by the 3′ to 5′ exonuclease activity of the Vent Polymerase, theconcentration of Vent polymerase may be titered to a level low enoughthat it will not degrade the second-stage primers (See Barnes WM. PNASUSA. 1994 Mar. 15; 91(6):2216-20. PMID: 8134376, which describes the useof a mixture of KlenTaq polymerase and Vent DNA polymerase where theKlenTaq comprises a major component of the mixture and Vent DNApolymerase is a minor component of the mixture). It is also possible touse second-stage primers that contain a few bases with phosphorthioatebonds at the 3′ ends of the oligonucleotide because these bases areresistant to digestion by the exo+ activity of 3′ to 5′ exonuclease DNApolymerases (See Chrisey, L. A. (1991) Antisense, Research andDevelopment 1 (1), 65-113; Spitzer, S.; Eckstein, F. (1988) Nucl. Acids.Res. 16 (24), 11691-11704. Connolly, B. A.; Potter, B. V. L.; Eckstein,F.; Pingoud, A.; Grotjahn, L. (1984) Biochemistry 23, 3443-3453; Stein,C. A.; Pal, R.; Devico, A. L.; Hoke, G.; Mumbauer, S.; Kinstler, O.;Sarngadharan, M. G.; Letsinger, R. L. (1991) Biochemistry 30 (9),2439-2444. Sayers, J. R.; Olsen, D. B.; Eckstein, F. (1989) Nucl. AcidsRes. 17 (22) 9495. Manson, J.; Brown, T.; Duff, G. (1990) LymphokineResearch 9 (1), 35-42. Woolf, T. M.; Jennings, C. B. G.; Rebagliati, M.;Melton, D. A. (1990) Nucl. Acids Res. 18 (7), 1763-1769. Leiter, J. M.E.; Agrawal, S.; Palese, P.; Zamecnik, P. C. (1990) Proc. Natl. Acad.Sci. USA 87 (9), 3430-3434. Reed, J. C.; Stein, C.; Subasinghe, C.;Haldar, S.; Croce, C. M.; Yum, S.; Cohen, J. (1990) Cancer Research 50(20), 6565-6570; Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L.(1990) J. Am. Chem Soc. 112, 1254-1255. Iyer, R. P.; Phillips, L. R.;Egan, W.; Regan, J. B.; Beaucage, S. L. (1990) J. Org. Chem. 55,4693-4699; Dagle, J. M.; Weeks, D. L.; Walder, J. A. (1991) Antisense,Research and Development 1 (1), 11-20. Maher, L. J.; Dolnick, B. J.(1988) Nucl. Acids Res. 16, 3341-3358. Walkder, R. Y.; Walkder, J. A.(1988) Proc. Natl. Acad. Sci. USA 85, 5011-5015, herein incorporated byreference in their entirety).

In addition to reducing the complexity of the second-stage PCR reaction,an exo+ DNA polymerase can also be used to convert longer first-stagePCR amplicons to shorter second-stage PCR amplicons that contain, attheir 3′ end, the complement of the universal primer sequence used forsequencing.

In some embodiments, certain sequencing methods (e.g., Illumina SBS orIon Torrent) determine sequencing reads from a single clonal population.It is understood that single nucleotide polymorphisms (SNPs) andinsertions and deletions (In/Dels) do not cause ambiguities in thesesequences that are determined from a single clonal population. Thus, bycomparing sequences across multiple sequence reads, SNPs and/or In/Delscan be located in the population of molecules sequenced, and thespecific changes in the SNP and In/Del then can be determined. In otherembodiments, where the sequences are not determined from a single clonalpopulation, SNPs and In/Dels may cause ambiguities in the sequencesbecause multiple populations of molecules may contribute to the sequencesignal. In some cases, these ambiguities in the sequences can arise forthe same reasons in conventional Sanger sequencing. As with thattechnique it can be possible to determine the nature of thepolymorphism, in that an SNP will show up as two different bases at oneparticular position. Similarly, a one base insertion will be evident asa single sequence up to that base position, and thereafter a mixture oftwo sequences that are a one base frame shift of each other. It can bepossible to deconvolute this information to determine SNPs and In/Dels,just as it has been done for Sanger sequencing.

Returning to FIG. 4, the illustrative array 681 may have between 100 and1000 spots, but greater or fewer spots are possible, in some cases thespots illustratively may be 2 mm in diameter with the overall arrayillustratively 25 mm×25 mm However, this size is illustrative only andany number of spots and any size array are contemplated. In oneembodiment, the spots on this array would contain both of a pair ofinner primers for a given amplicon generated in first-stageamplification, illustratively in blister 664. The inner primersillustratively each have on their 5′ ends an additional sequence that iseither complementary to a universal forward or a universal reverseprimer (e.g., similar to the common sequencing primers introduced duringlibrary production in several different NGS methods). In one embodiment,a barcode primer sequence, as is commonly used in next generationsequencing, may be omitted because the location of the spot encodes theinformation about the amplicon present in that spot and therefore theidentity of the sequence that will be generated in that spot.

After a number of cycles (between about 10 and about 26 cycles dependingon the specific assay, wherein 10 cycles may be sufficient for abundantpathogens, and 26 cycles for less abundant targets to get to saturationof product formation in first-stage PCR) this first-stage PCR materialis flooded into array 681, as described above. In one example, thesample is transferred to array 681 while hot (so that the PCR productsare denatured) and is allowed to cool and hybridize to the primers onarray 681. DNA polymerase and dNTPs that enter second-stageamplification zone 680 can extend the tethered oligonucleotides that arehybridized. Illustratively, first-stage amplification product may bediluted or may be used without dilution, as sequencing can be performedafter a single round of extension off of the tethered oligonucleotide onthe array. After extension, extra material can be pushed back throughchannel 665 or out through channel 667, and new PCR buffer, enzyme anddNTPs may be introduced from any or all of entry wells 615 i, 615 j, and615 k. This removes all solution amplicon and the first-stage primers.

As above, subsequent to amplification in blister 664, amplification maybe performed in second-stage amplification zone 680, by moving amplicon221 into zone 680. Amplification on array 681 using a tethered primer224 and reverse primer optionally with a sequence complementary to theuniversal primer as described above. FIGS. 5A-5C show an example usingbridge PCR. Amplification of first-stage amplicon 221 first occurs usingthe S_(iFx) portion of forward primer 224 (FIG. 5A (i)-(iii)) togenerate second-stage amplicon 229. The reverse strand 230 may begenerated using reverse primer S_(iRx) (FIG. 5B (iv)-(v)). An exo+ DNApolymerase can be used to repair an end 234 of long first-stage ampliconso that it has the U_(R) primer sequence at its 3′ end (FIG. 5C (vi) and(vii)) to be available for sequencing using a U_(R) primer. Strand 230includes the complement of the universal primer, U_(F), at its 3′end sothat strand 230 optionally can also be sequences using universal primerU_(F) using methods similar to those used on the MiSeq or HiSeq(Illumina). Optionally, either or both strands 229, 230 may be sequencedin this embodiment.

Bridge PCR can be done in the presence of a dsDNA binding dye such asLCGreen® (BioFire Diagnostics, LLC), or in the presence of probes orother detecting methods as are known in the art. In one illustrativeembodiment, subsequent to bridge PCR, the array is melted andfluorescence from the dsDNA binding dye is used to generate a meltingcurve that can indicate the presence of specifically amplified products.If the reaction completely failed, then there will be no melting curveand the process may not proceed to the sequencing reaction.

If there is amplicon on the array, then channels 665 and 667 may be usedfor introducing and removing solutions for sequencing. Then sequencingcan be performed by any conventional method, including those describedin this application, including by methods such as those described byIllumina (See, e.g., U.S. Pat. Nos. 7,544,794 and 8,946,397, hereinincorporated by reference).

It understood that other sequencing mechanisms may also be used. Forexample an embodiment similar to the 454 pyrosequencing technology maybe employed, wherein dNTPs incorporated at a particular spot 682generate pyrophosphate and additional enzymes convert this to ATP andthen to light. This light is detected to reveal the nucleotide sequence.Similarly it may be possible to use Ion Torrent-type technology wherethe signal generated is a proton and the detection is a pH sensitivetransistor element under the array.

Sequencing also could be performed by measuring current changes ofmolecules flowing through a Nanopore-style channel incorporated in array681, or array 681 may be moved to the Nanopore instrument. Othersequencing methods are known in the art and are contemplated herein.

In an alternate embodiment, the same pouch 610, as discussed above, maybe used, or other vessels for two-step PCR may be employed. However, inthis embodiment, only one inner primer of each primer pair is tetheredto the array and all of the inner primers are present in theamplification solution in second-stage amplification zone 680. Thisimplementation has the advantage that amplification in solution togetherwith amplification on the array is fast and efficient, but it also hasthe disadvantage that all of the inner primers are present in themixture so there will be more non-specific products made and this mayresult in the products bound to each spot not being a single molecularspecies. In this implementation, after amplification and melt analysis,the array may be washed of reverse strands and the array becomes like asequencing library, with the appropriate series of sequencing chemicalsflooded over the thin volume of the array and detection as describedabove. This embodiment is similar to that described with respect to FIG.1, except that amplification on array 682 is performed in a singlereaction mixture.

Illustratively, in a similar embodiment, only the specific inner forwardprimers (S_(iFx) are 324 are tethered to array 681 and only the reverseprimers 332 are present in solution (FIG. 6A) for further amplificationof first-stage amplicon 321. An advantage is that the multiplexsecond-stage reaction has roughly half the number of primers present,which should reduce non-specific amplification. However, it also meansthat the kinetics of amplification are slower since one strand is onlymade on the array and the other strand is only made in solution (FIG.6A-6B (i)-(iv)). In this embodiment, after amplification and optionalmelt analysis (FIGS. 6A-6B (i)-(iv)), the array is washed of reversestrands (subsequent to denaturation, FIG. 6B (iii)-(iv)) and the arrayfunctions as a sequencing library, with the appropriate series ofsequencing chemicals flooded over the thin volume of the array anddetection, as described above.

Moreover, non-specific amplification with the second-stage reverseprimers in solution about array 681 should not interfere with thegeneration of single molecular species of a second-stage amplicon ateach spot 682 on array 681 because the sequence complexity of thematerial entering this second-stage reaction has been reduced by theenrichment from first-stage PCR. However, it is understood thatadditional steps can be taken, together or separately, to ensure thesingle molecular species. In one such step, a 3′ to 5′ exo+ DNApolymerase can be used, as described above, to reduce the sequencecomplexity of the input material still further.

In another illustrative example, the specificity of PCR can be increasedusing methods such as the Templex method of Genaco Biomedical Products,Inc. (Han J. et al. JCM. 2006 November; 44(11):4157-62. PMID: 17005760).In this illustrative embodiment, the primers 332 present in solutionover array 681 include a Universal Reverse sequence 330 that binds onthe 5′ side of the specific inner primers (best seen in FIGS. 6C-6D).Illustratively, these primers are present at 1/10th to 1/100th of thenormal concentration present in a standard PCR (standard is 0.4 to 0.8μM). Creation of primer dimers requires a tri-molecular complex of twoprimers and DNA polymerase and the rate of formation of this complex isa function of the concentration of the primers. Thus if each of theprimers is present at 1/10^(th) of the concentration in a standard PCRthen the rate of primer dimer formation should be reduced to 1/100^(th).In addition to the U_(R)S_(iR) primers 332 present at low concentrationthere is an additional primer—the U_(R) sequence—present at standardprimer concentration. After a few cycles (illustratively 3 to 10 cycles)of PCR performed with long extension times so that the low concentrationU_(R)S_(iR) primers have time to hybridize to their targets (FIGS. 6C-6D(v)-(viii)), there is enough complement of the U_(R) sequence in thenascent amplicons 329. At this point, the U_(R)S_(iR) primers optionallycan be washed away and an exo+ DNA polymerase can be introduced torepair amplicon 329 to generate amplicon 329 a, so that it contains asequence complementary to the universal primer 320 (FIGS. 6D-6E(viii)-(ix)). Then a single U_(R) primer at the high concentration canbe introduced to anneal to this templates (FIG. 6E (xi)) and PCR cyclingcan proceed with standard annealing and extension times. The net resultis that the specificity of the second-stage PCR is increased and thuseach feature on the array is more likely to be a single molecularspecies. Thus, increased specificity is obtained in exchange for a fewslower cycles in the early part of PCR.

In a third example, a further extension of the Templex method uses the“self-avoiding molecular recognition system” (SAMRS) and the“artificially expanded genetic information system” (AEGIS) of Benner(Glushakova et al. J. Virol. Methods. 2015 March; 214:60-74. PMID:25680538), herein incorporated by reference. This enables clean highlevel multiplex PCR amplification in nested PCR formats. This can beimplemented directly as described in the second-stage multiplex reactionin the second-stage reaction zone 680.

Example 4

Sequencing multiple regions nested within a first-stage PCR amplicon isnow described. In some situations it will be advantageous to determinethe sequence of many regions of the first-stage PCR amplicon,illustratively derived from different second-stage amplicons nestedwithin the first-stage PCR target. For example, mutations in TEM betalactamase that confer the ESBL phenotype can be spread across the gene.One can amplify a large portion of the gene in the first-stageamplification reaction, and then amplify various smaller regions insecond-stage PCR.

In one illustrative example, overlapping amplicons are generated insecond-stage PCR. When performed in discrete reactions, such as in wells582 of the device of FIG. 1, these overlapping amplicons can be easilygenerated. In the array 681 of FIG. 4, the specificity of theamplification comes from the primers attached to the array spot 682.However, the primers in solution and the primers on the spots 682 mayoverlap (see FIG. 7A (i)-(ii)) in such a manner that instead of making adiscrete set of overlapping second-stage amplicons (I1 to IS in FIG.7A(i)), even shorter amplicons may be generated (for example betweenS_(iFb1) and S_(iRa1), which would generate an amplicon that is only theportion of I1 and I2 that overlaps) that do not overlap and that do notcompletely cover the sequence.

This problem may be avoided by splitting the second-stage PCR into twocompartments, both similar to second-stage amplification zone 680, withtwo sets of reactions (FIGS. 7B and 7C). In this implementation theamplicons generated in second-stage A (FIG. 7B (iv)) are not overlappingso that undesired short amplicons are minimized or eliminated. Theintervening amplicons are generated in second-stage B (FIG. 7B (v)), andthey similarly are not overlapping, so that the undesired shortamplicons are similarly minimized or eliminated.

Example 5

This implementation is similar to Example 3 above, except that all ofthe inner primers are present in the mixture (FIG. 8A). This has theadvantage that amplification in solution together with amplification onthe array is fast and efficient (FIG. 8B). To add an optional filter forspecificity of the single molecular species that is synthesized on array681, the capture primers may be “inner-inners”. The inner-innersS_(iiFx) hybridize to a sequence internal to the solution-based innerprimers S_(iFx), which is internal to the outer primers S_(oFx) (seeFIG. 8A (i)). The inner-inner primer S_(iiFx) can partially overlap withthe inner primer S_(iFx) in that direction as long as a few bases(illustratively at least 4 bases) are internal to (or 3′ of) the S_(iFx)sequence. Solution-based PCR takes place using the inner primersS_(iFn and) U_(R)S_(iRn), as shown in FIG. 8B (iv), and simultaneouslyon array 682, as shown in FIG. 8B (iii), resulting in a tetheredamplicon having the appropriate universal primer, as shown in FIG. 8C(v). In this embodiment, because added specificity is provided using thetwo sets of nested primers S_(iFx) and S_(iiFx), it is understood thatfirst-stage amplification with S_(oFx) is optional, and that allamplification may take place in a single amplification chamber.Preparing array 681 for sequencing may be according to any of themethods discussed above.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Whilecertain embodiments and details have been included herein and in theattached invention disclosure for purposes of illustrating theinvention, it will be apparent to those skilled in the art that variouschanges in the methods and apparatus disclosed herein may be madewithout departing from the scope of the invention, which is defined inthe appended claims. All changes which come within the meaning and rangeof equivalency of the claims are to be embraced within their scope.

1.-36. (canceled)
 37. A container for amplification and sequencing of aplurality of nucleic acids, the container comprising: a first-stageamplification chamber, the first-stage amplification chamber comprisinga plurality of first-stage pairs of primers, each first-stage pair ofprimers configured for amplification of one of the plurality of nucleicacids to generate a plurality of first-stage amplicons; and asecond-stage amplification chamber, the second-stage amplificationchamber comprising a plurality of second-stage primer pairs, eachsecond-stage primer pairs configured for amplification of at least aportion of a sequence of one of the first stage-amplicons to generatesecond-stage amplicons, wherein the second-stage amplification chamberis further configured for sequencing the second-stage amplicons.
 38. Thecontainer of claim 37, wherein at least one member of each of thesecond-stage primer pairs is nested within a corresponding first-stageprimer.
 39. The container of claim 37, wherein at least one member ofeach of the second-stage primer pairs is tethered to a support.
 40. Thecontainer of claim 39, wherein the at least one member of each of thesecond-stage primer pairs is tethered to the support by the 5′ end. 41.The container of claim 37, wherein the second-stage amplicons comprisesingle molecular species.
 42. The container of claim 37, furthercomprising one or more sealable ports, the one or more sealable portsproviding the only access from an exterior of the container to thefirst-stage amplification chamber and the second-stage amplificationchamber, such that when the one or more sealable ports are sealed, thecontainer is fully closed.
 43. The container of claim 37, wherein thesecond-stage amplification chamber comprises an inlet channel and anoutlet channel and opening and closing the inlet channel and the outletchannel controls flow of fluid across the second-stage amplificationchamber.
 44. The container of claim 37, further comprising a lysischamber configured to receive a sample and to prepare a lysed sample.45. The container of claim 44, further comprising a nucleic acidextraction chamber configured to receive the lysed sample and to extractnucleic acids from the lysed sample.
 46. The container of claim 37,wherein the second-stage amplification chamber comprises an array ofwells, each well configured to carry out a second-stage amplificationreaction.
 47. The container of claim 37, wherein the second-stageamplification chamber comprises an array of spots, each spot comprisingone of the second-stage primer pairs, and each spot configured to carryout a second-stage amplification reaction.
 48. The container of claim47, wherein for each spot at least one primer of the second-stage primerpair is tethered to the spot.
 49. The container of claim 47, wherein thenumber of spots totals no more than 512 spots.
 50. The container ofclaim 47, wherein the number of spots totals no more than 256 spots.51.-55. (canceled)
 56. A container for amplifying and sequencing nucleicacids, the container comprising: a first chamber comprising a pluralityof first primer pairs, each first primer pair configured to amplify oneof the nucleic acids to generate a plurality of first amplicons; and asecond chamber comprising a plurality of second primer pairs, eachsecond primer pair configured to amplify at least a portion of asequence of one of the first amplicons to generate second amplicons;wherein the second chamber is further configured for sequencing thesecond amplicons.
 57. The container of claim 56, wherein at least onemember of each of the second primer pairs is nested within acorresponding first primer.
 58. The container of claim 56, wherein thesecond amplicons comprise single molecular species.
 59. The container ofclaim 56, further comprising one or more sealable ports providing theonly access from an exterior of the container to the first chamber andthe second chamber such that when the one or more sealable ports aresealed, the container is fully closed.
 60. The container of claim 56,further comprising a lysis chamber configured to receive a sample and toprepare a lysed sample.
 61. The container of claim 60, furthercomprising a nucleic acid extraction chamber configured to receive thelysed sample and to extract nucleic acids from the lysed sample.